Isolated tumor rejection antigen precursor MAGE-2 derived peptides, and uses thereof

ABSTRACT

The invention describes peptides derived from tumor rejection antigen precursor MAGE-2. These peptides bind with HLA-A2 molecules, thus presenting complexes which provoke cytolytic T cell production. The resulting &#34;CTLs&#34; are specific for complexes of HLA-A2 and the peptide. The complexes can be used to generate monoclonal antibodies. The cytolytic T cells produced may be used in the context of immunotherapy, such as adoptive transfer.

This application is a Divisional of Ser. No. 08/217,188 filed Mar. 24,1994, now U.S. Pat. No. 5,554,724.

FIELD OF THE INVENTION

This invention relates to immunogenetics and to peptide chemistry. Moreparticularly, it relates to peptides, especially undecapeptites,decapeptides, and nonapeptides useful in various ways, includingimmunogens and as ligands for the HLA-A2 molecule. More particularly, itrelates to a so-called "tumor rejection antigen", derived from the tumorrejection antigen precursor encoded by gene MAGE-2, and presented by theMHC-class I molecule HLA-A2.

BACKGROUND AND PRIOR ART

The study of the recognition or lack of recognition of cancer cells by ahost organism has proceeded in many different directions. Understandingof the field presumes some understanding of both basic immunology andoncology.

Early research on mouse tumors revealed that these displayed moleculeswhich led to rejection of tumor cells when transplanted into syngeneicanimals. These molecules are "recognized" by T-cells in the recipientanimal, and provoke a cytolytic T-cell response with lysis of thetransplanted cells. This evidence was first obtained with tumors inducedin vitro by chemical carcinogens, such as methylcholanthrene. Theantigens expressed by the tumors and which elicited the T-cell responsewere found to be different for each tumor. See Prehn, et al., J. Natl.Canc. Inst. 18: 769-778 (1957); Klein et al., Cancer Res. 20: 1561-1572(1960); Gross, Cancer Res. 3: 326-333 (1943), Basombrio, Cancer Res. 30:2458-2462 (1970) for general teachings on inducing tumors with chemicalcarcinogens and differences in cell surface antigens. This class ofantigens has come to be known as "tumor specific transplantationantigens" or "TSTAs". Following the observation of the presentation ofsuch antigens when induced by chemical carcinogens, similar results wereobtained when tumors were induced in vitro via ultraviolet radiation.See Kripke, J. Natl. Canc. Inst. 53: 333-1336 (1974).

While T-cell mediated immune responses were observed for the types oftumor described supra, spontaneous tumors were thought to be generallynon-immunogenic. These were therefore believed not to present antigenswhich provoked a response to the tumor in the tumor carrying subject.See Hewitt, et al., Brit. J. Cancer 33: 241-259 (1976).

The family of tum⁻ antigen presenting cell lines are immunogenicvariants obtained by mutagenesis of mouse tumor cells or cell lines, asdescribed by Boon et al., J. Exp. Med. 152: 1184-1193 (1980), thedisclosure of which is incorporated by reference. To elaborate, tum⁻antigens are obtained by mutating tumor cells which do not generate animmune response in syngeneic mice and will form tumors (i.e., "tum⁺ "cells). When these tum⁺ cells are mutagenized, they are rejected bysyngeneic mice, and fail to form tumors (thus "tum⁻ "). See Boon et al.,Proc. Natl. Acad. Sci. USA 74: 272 (1977), the disclosure of which isincorporated by reference. Many tumor types have been shown to exhibitthis phenomenon. See, e.g., Frost et al., Cancer Res. 43: 125 (1983).

It appears that tum⁻ variants fail to form progressive tumors becausethey initiate an immune rejection process. The evidence in favor of thishypothesis includes the ability of "tum⁻ " variants of tumors, i.e.,those which do not normally form tumors, to do so in mice with immunesystems suppressed by sublethal irradiation, Van Pel et al., Proc. Natl.Acad. Sci. USA 76: 5282-5285 (1979); and the observation thatintraperitoneally injected tum⁻ cells of mastocytoma P815 multiplyexponentially for 12-15 days, and then are eliminated in only a few daysin the midst of an influx of lymphocytes and macrophages (Uyttenhove etal., J. Exp. Med. 152: 1175-1183 (1980)). Further evidence includes theobservation that mice acquire an immune memory which permits them toresist subsequent challenge to the same tum⁻ variant, even whenimmunosuppressive amounts of radiation are administered with thefollowing challenge of cells (Boon et al., Proc. Natl, Acad. Sci. USA74: 272-275 (1977); Van Pel et al., supra; Uyttenhove et al., supra).

Later research found that when spontaneous tumors were subjected tomutagenesis, immunogenic variants were produced which did generate aresponse. Indeed, these variants were able to elicit an immuneprotective response against the original tumor. See Van Pel et al., J.Exp. Med. 157: 1992-2001 (1983). Thus, it has been shown that it ispossible to elicit presentation of a so-called "tumor rejection antigen"in a tumor which is a target for a syngeneic rejection response. Similarresults have been obtained when foreign genes have been transfected intospontaneous tumors. See Fearon et al., Cancer Res. 48: 2975-1980 (1988)in this regard.

A class of antigens has been recognized which are presented on thesurface of tumor cells and are recognized by cytolytic T cells, leadingto lysis. This class of antigens will be referred to as "tumor rejectionantigens" or "TRAs" hereafter. TRAs may or may not elicit antibodyresponses. The extent to which these antigens have been studied, hasbeen via cytolytic T cell characterization studies, in vitro i.e., thestudy of the identification of the antigen by a particular cytolytic Tcell ("CTL" hereafter) subset. The subset proliferates upon recognitionof the presented tumor rejection antigen, and the cells presenting theantigen are lysed. Characterization studies have identified CTL cloneswhich specifically lyse cells expressing the antigens. Examples of thiswork may be found in Levy et al., Adv. Cancer Res. 24: 1-59 (1977); Boonet al., J. Exp. Med. 152: 1184-1193 (1980); Brunner et al., J. Immunol.124: 1627-1634 (1980); Maryanski et al., Eur. J. Immunol. 124: 1627-1634(1980); Maryanski et al., Eur. J. Immunol. 12: 406-412 (1982); Palladinoet al., Canc. Res. 47: 5074-5079 (1987). This type of analysis isrequired for other types of antigens recognized by CTLs, including minorhistocompatibility antigens, the male specific H-Y antigens, and theclass of antigens referred to as "tum-" antigens, and discussed herein.

A tumor exemplary of the subject matter described supra is known asP815. See DePlaen et al., Proc. Natl. Acad. Sci. USA 85: 2274-2278(1988); Szikora et al., EMBO J 9: 1041-1050 (1990), and Sibille et al.,J. Exp. Med. 172: 35-45 (1990), the disclosures of which areincorporated by reference. The P815 tumor is a mastocytoma, induced in aDBA/2 mouse with methylcholanthrene and cultured as both an in vitrotumor and a cell line. The P815 line has generated many tum⁻ variantsfollowing mutagenesis, including variants referred to as P91A (DePlaen,supra), 35B (Szikora, supra), and P198 (Sibille, supra). In contrast totumor rejection antigens--and this is a key distinction--the tum⁻antigens are only present after the tumor cells are mutagenized. Tumorrejection antigens are present on cells of a given tumor withoutmutagenesis. Hence, with reference to the literature, a cell line can betum⁺, such as the line referred to as "P1", and can be provoked toproduce tum⁻ variants. Since the tum⁻ phenotype differs from that of theparent cell line, one expects a difference in the DNA of tum⁻ cell linesas compared to their tum⁺ parental lines, and this difference can beexploited to locate the gene of interest in tum⁻ cells. As a result, itwas found that genes of tum⁻ variants such as P91A, 35B and P198 differfrom their normal alleles by point mutations in the coding regions ofthe gene. See Szikora and Sibille, supra, and Lurquin et al., Cell 58:293-303 (1989). This has proved not to be the case with the TRAs of thisinvention. These papers also demonstrated that peptides derived from thetum⁻ antigen are presented by the L^(d) molecule for recognition byCTLs. P91A is presented by L^(d), P35 by D^(d) and P198 by K^(d).

PCT application PCT/US92/04354, filed on May 22, 1992 assigned to thesame assignee as the subject application, teaches a family of humantumor rejection antigen precursor coding genes, referred to as the MAGEfamily. Several of these genes are also discussed in van der Bruggen etal., Science 254: 1643 (1991). It is now clear that the various genes ofthe MAGE family are expressed in tumor cells, and can serve as markersfor the diagnosis of such tumors, as well as for other purposesdiscussed therein. See also Traversari et al., Immunogenetics 35: 145(1992); van der Bruggen et al., Science 254: 1643 (1991). The mechanismby which a protein is processed and presented on a cell surface has nowbeen fairly well documented. A cursory review of the development of thefield may be found in Barinaga, "Getting Some `Backbone`: How MHC BindsPeptides", Science 257: 880 (1992); also, see Fremont et al., Science257: 919 (1992); Matsumura et al., Science 257: 927 (1992); Latron etal., Science 257: 964 (1992). These papers generally point to arequirement that the peptide which binds to an MHC/HLA molecule be nineamino acids long (a "nonapeptide"), and to the importance of the firstand ninth residues of the nonapeptide.

Studies on the MAGE family of genes have now revealed that a particularnonapeptide is in fact presented on the surface of some tumor cells, andthat the presentation of the nonapeptide requires that the presentingmolecule be HLA-A1. Complexes of the MAGE-1 tumor rejection antigen (the"TRA" or nonapeptide") leads to lysis of the cell presenting it bycytolytic T cells ("CTLs").

Attention is drawn to U.S. Pat. No. 5,554, 506 to Traversari, et al. andU.S. Pat. No. 5,585,451 to Townsend, et al., both of which present workon other, MAGE-derived peptides.

Research presented in, e.g., U.S. patent application Ser. No. 07/938,334filed Aug. 31, 1992, now U.S. Pat. No. 5,405,940 and in U.S. patentapplication Ser. No. 073,103, filed Jun. 7, 1993, found now U.S. Pat.No. 5,462,871 when comparing homologous regions of various MAGE genes tothe region of the MAGE-1 gene coding for the relevant nonapeptide, thereis a great deal of homology. Indeed, these observations lead to one ofthe aspects of the invention disclosed and claimed therein, which is afamily of nonapeptides all of which have the same N-terminal andC-terminal amino acids. These nonapeptides were described as beinguseful for various purposes which includes their use as immunogens,either alone or coupled to carrier peptides. Nonapeptides are ofsufficient size to constitute an antigenic epitope, and the antibodiesgenerated thereto were described as being useful for identifying thenonapeptide, either as it exists alone, or as part of a largerpolypeptide.

These references, especially U.S. Pat. No. 5,462,871, showed aconnection between HLA-A1 and MAGE-3; however, only about 26% of thecaucasian population and 17% of the negroid population presents HLA-A1molecules on cell surfaces. Thus, it would be useful to have additionalinformation on peptides presented by other types of MHC molecules, sothat appropriate portions of the population may benefit from theresearch discussed supra.

It has now been found that antigen presentation of MAGE-2 derivedpeptides set forth, in the disclosure which follows, identifies peptideswhich complex with MHC class I molecule HLA-A2. The ramifications ofthis discovery, which include therapeutic and diagnostic uses, are amongthe subjects of the invention, set forth in the disclosure whichfollows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an exemplary graph showing the calculation of peptide (SEQ IDNO:87) concentration which includes 0.5 maximum upregulation ofHLA-A2.1.

FIG. 2 presents comparative data on the response of HPV clones tovarious materials, as measured by ⁵¹ Cr release assays.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1

Experimental Conditions:

All experiments were performed at room temperature unless statedotherwise. All Fmoc protected aminoacids, synthesis polymers, peptidesand TFA were stored at -20° C.

Peptide Synthesis

Peptides were synthesized by solid phase strategies on an automatedmultiple peptide synthesizer (Abimed AMS 422) (see Gausepohl and FrankBiotech, September 1990; Gausepohl etal., in E. Giralt and D. Andreu,(eds). Peptides 1990: 206-207 (1990).

The peptides were made in various runs, in each of which 48 differentpeptides were synthesized simultaneously.

Tentagel S AC (Rapp et al., In Innovation and Perspectives in SolidPhase Peptide Synthesis, 205-210 (1990); Sheppard and Williams, Int. J.Peptide Protein Res. 20: 451-454 (1982), a graft polymer ofpolyethyleneglycol spacer arms on a polystyrene matrix, was used as aresin (40-60 mg per peptide, 10 μmol Fmoc amino acid loading).

Repetitive couplings were performed by adding a mixture of 90 μl 0.67 MBOP Gausepohl et al., Peptides 1988: 241-243 (1988); Castro et al., Tet.Lett 14: 1219-1222 (1975)) in NMP, 20 μl NMM in NMP 2/1 (v/v) and 100 μlof an 0.60 M solution of the appropriate Fmoc amino acid (Fields andNoble, Int. J. Pep. Prot. Res. 35: 161-214 (1990) in NMP (6-fold excess)to each reaction vessel. At 70% of the reaction time approximately 50 μldichloromethane was added to each reaction vessel.

Fmoc-deprotection was performed by adding 3 times 0.8 ml ofpiperidine/DMA 1/4 (v/v) to each reaction vessel.

Coupling- and deprotection times were increased as the synthesisproceeded, starting with 30 min and 3 times 3 min respectively.

Washings after couplings and Fmoc-deprotections were done with 6 times1.2 ml DMA. After the required sequence had been reached and the lastFmoc-protection was removed the peptidylresin was washed extensivelywith DMA, dichloromethane, dichloromethane/ ether 1/1 (v/v) and etherrespectively, and dried.

Peptide Cleavage and Isolation

Cleavage of the peptides from the resin and removal of the side chainprotecting groups was performed by adding 6 times 200 μl TFA/water 19/1(v/v) at 5 min intervals to each reaction vessel, thus yielding freecarboxylic peptides. For Trp-containing peptides TFA/water/ethanethiol18/1/1 (v/v/v) was used.

Two hours after the first TFA addition the peptides were precipitatedfrom the combined filtrates by addition of 10 ml ether/pentane 1/1 (v/v)and cooling to -20° C. The peptides were isolated by centrifugation(-20° C., 2500 g, 10 min).

After treatment of the pellet with ether/pentane 1/1 (v/v) and isolationby the same centrifugation procedure, the peptides were dried at 45° C.for 15 min.

Each of the peptides was dissolved in 2 ml water (or 2 ml 10 vol. %acetic acid), the solution frozen in liquid nitrogen for 3 min, andlyophilized while being centrifuged (1300 rpm, 8-16 h).

Analysis and Purification

The purity of the peptides was determined by reversed phase HPLC; analiquot of about 50 nmol was dissolved in 100 μl 30 vol. % acetic acid.Of this solution 30 μl was applied to an RP-HPLC system equipped with aternary solvent system; A: water, B: acetonitrile, C: 2 vol. % TFA inwater.

Gradient elution (1.0 ml/min) was performed from 90% A, 5% B, 5% C to20% A, 75% B, 5%C in 30 min. Detection was at 214 nm.

Samples taken at random were analyzed by mass spectrometry on a PDMS.The 31 binding peptides were all analyzed by mass spectrometry on a PDMSand by quantative amino acid analysis after hydrolysis on a BPAminoquant. Of all analyzed samples the difference between calculatedand measured masses was within the experimental error (0.1%) asspecified by the producer of the equipment used. All amino acidcompositions were as expected.

EXAMPLE 2

Peptides

Of all 71 MAGE-2 peptides that had been freeze dried, 1 mg was weighedand dissolved in 10 μl of DMSO. Of all dissolved peptides a dilution of0.5 mg/ml in 0.9% NaCl was made and the pH was neutralized to pH 7 with5% acetic acid diluted in distilled water (CE₃ COOH, Merck Darmstadt,Germanys:) or 1N NaOH diluted in distilled water (Merck Darmstadt,Germany:).

Cells

174CEM.T2 cells were cultured in Iscove's modified Dulbecco's medium(Biochrom KG Seromed Berlin, Germany: F0465) supplemented with 100IU/mlpenicillin (Biocades Pharma, Leiderdorp, The Netherlands), 100 μg/mlkanamycin (Sigma St. Louis, USA), 2 mM glutamine (ICN Biomedicals Inc.Costa Mesa, Calif., USA:) and 10% fetal calf serum (FCS, HycloneLaboratories Inc. Logan, Utah, USA:). Cells were cultured at a densityof 2.5×10⁵ /ml during 3 days at 37° C., 5% CO₂ in humified air.

Peptide Binding

174CEM.T2 cells were washed twice in culture medium without FCS and putin serum-free culture medium to a density of 2×10⁶ cells/ml. Of thissuspension 40 μl was put into a V bottomed 96 well plate (Greiner GmbH,Frickenhausen, Germany:) together with 10 μl of twofold serial dilutionsin 0.9% NaCl of the individual peptide dilutions (ranging from 500 μg/mlto 15.6 μg/ml). The end concentrations range from 200 μg/ml to 3.1 μg/mlpeptide with 8×10⁴ 174CEM.T2 cells. This solution was gently agitatedfor 3 minutes after which an incubation time of 16 hours at 37° C., 5%CO₂ in humified air took place. Then cells were washed once with 100 μl,0.9% NaCl, 0.5% bovine serum albumin (Sigma St. Louis, USA:), 0.02% NaN₃(Merck Darmstadt, Germany:822335). After a centrifuge round of 1200 rpmthe pellet was resuspended in 50 μl of saturating amounts of HLA-A2.1specific mouse monoclonal antibody BB7.2 for 30 minutes at 4° C. Thencells were washed twice and incubated for 30 minutes with F(ab)₂fragments of goat anti-mouse IgG that had been conjugated withfluorescein isothiocyanate (Tago Inc Burlingame, Calif., USA:) in adilution of 1:40 and a total volume of 25 μl.

After the last incubation, cells were washed twice and fluorescence wasmeasured at 488 nanometer on a FACScan flow-cytometer (Becton Dickinson,Franklin Lakes, N.J., USA).The concentration at which the 0.5 maximumupregulation of HLA-A2.1 on 174CEM.T2 cells was achieved was determinedusing graphs in which the fluorescence index was plotted against thepeptide concentration. The results are shown in Table I.

                  TABLE I                                                         ______________________________________                                        Binding affinities of peptides derived from human melanoma                      associated protein MAGE-2 that fit the HLA-A2.1 motif                         SEQ.                          peptide concentra-                              ID.   tion that induces                                                       NO. sequence residues 0.5 maximum FI                                        ______________________________________                                        12     GLEARGEALGL   15-25    >100   μg/ml                                   13 GLEARGEAL 15-23 60 μg/ml4                                               14 ALGLVGAQA 22-30 >100 μg/ml                                              15 GLVGAQAPA 24-32 65 μg/ml                                                16 DLESEFQAA 100-108 >100 μg/ml                                            17 DLESEFQAAI 100-109 >100 μg/ml                                           18 AISRKMVELV 108-117 >100 μg/ml                                           19 AISRKMVEL 108-116 >100 μg/ml                                            3 KMVELVHFL 112-120 40 μg/ml                                               20 KMVELVHFLL 112-121 >100 μg/ml                                           21 KMVELVHFLLL 112-122 >100 μg/ml                                          22 LLLKYRAREPV 120-130 >100 μg/ml                                          23 LLKYRAREPV 121-130 >100 μg/ml                                           24 VLRNCQDFFPV 139-149 >100 μg/ml                                          4 VIFSKASEYL 149-158 35 μg/ml                                              5 YLQLVFGIEV 157-166 35 μg/ml                                              25 YLQLVFGIEVV 157-167 >100 μg/ml                                          6 QLVFGIEVV 159-167 25 μg/ml                                               7 QLVFGIEVVEV 159-169 30 μg/ml                                             26 GIEVVEVVPI 163-172 >100 μg/ml                                           27 PISHLYILV 171-179 55 μg/ml                                              28 HLYILVTCL 174-182 >100 μg/ml                                            29 HLYILVTCLGL 174-184 >100 μg/ml                                          30 YILVTCLGL 176-184 >100 μg/ml                                            31 CLGLSYDGL 181-189 65 μg/ml                                              32 CLGLSYDGLL 181-190 >100 μg/ml                                           33 VMPKTGLLI 195-203 >100 μg/ml                                            34 VMPKTGLLII 195-204 >100 μg/ml                                           35 VMPKTGLLIIV 195-205 >100 μg/ml                                          36 GLLIIVLAI 200-208 >100 μg/ml                                            37 GLLIIVLAII 200-209 >100 μg/ml                                           38 GLLIIVLAIIA 200-210 >100 μg/ml                                          39 LLIIVLAII 201-209 >100 μg/ml                                            40 LLIIVLAIIA 201-210 >100 μg/ml                                           41 LLIIVLAIIAI 201-211 >100 μg/ml                                          42 LIIVLAIIA 202-210 >100 μg/ml                                            43 LIIVLAIIAI 202-211 >100 μg/ml                                           8 IIVLAIIAI 203-211 20 μg/ml                                               44 IIAIEGDCA 208-216 >100 μg/ml                                            45 KIWEELSML 220-228 >100 μg/ml                                            9 KIWEELSMLEV 220-230 25 μg/ml                                             46 LMQDLVQENYL 246-256 >100 μg/ml                                          47 FLWGPRALI 271-279 65 μg/ml                                              10 ALIETSYVKV 277-286 20 μg/ml                                             49 ALIETSYVKVL 277-287 >100 μg/ml                                          11 LIETSYVKV 278-286 30 μg/ml                                              63 LIETSYVKVL 278-287 55 μg/ml                                             50 TLKIGGEPHI 290-299 >100 μg/ml                                           51 HISYPPLHERA 298-308 >100 μg/ml                                        ______________________________________                                    

The 174CEM.T2 cell line expresses "empty" and unstable HLA-A2.1molecules that can be stabilized when a peptide is binding to thepeptide presenting groove of these molecules. A stabilized HLA-A2.1molecule that will not easily degrade is the result of binding of ananalyzed peptide. This leads to an increase in cell surface expressionof the HLA-A2.1 molecule. The fluorescence index is a measure for theamount of upregulation of HLA-A2.1 molecules. This fluorescence index iscalculated according to the following formula:

MF=Mean Fluorescence ##EQU1## Fluorescence Index of the backgroundfluorescence is 0. Results

In order to identify MAGE-2 peptides that could bind to HLA-A2.1molecules expressed by 174CEM.T2 cells, the amino acid sequence ofMAGE-2 was examined in accordance with van der Bruggen et al., Science254: 1643-1647 (1991). All peptides of nine, ten or eleven amino acidsthat fitted the published HLA-A2.1 binding motif were examined (TableI).

Only the peptides of SEQ ID NOS: 1-11 of Table III were able toupregulate the expression of HLA-A2.1 molecules at low peptideconcentration, indicating their binding to the HLA-A2.1 molecule asdescribed in Example 2. None of the 50 other peptides were able to dothis. The results of the fluorescence measurement are given in Tables Iand II. The 0.5 maximum upregulation of HLA-A2.1 molecules on 174CEM.T2cells was determined using graphs in which the FI was plotted againstthe peptide concentration for each individual peptide.

These experiments indicate that only a limited proportion of peptidesthat fit the HLA-A2.1 motif have the ability to bind to this HLAmolecule with high affinity and are therefore candidates of the MAGE-2protein to be recognized by human CTL, which recognize peptides onlywhen bound to HLA molecules.

                  TABLE II                                                        ______________________________________                                        Binding affinities of additional peptides derived from human                    melanoma associated protein MAGE-2 that fit the extended HLA-                 A2.1 motif (Ruppert et al cell 74: 929-937 (1993)).                                                         peptide concentra-                              SEQ.   tion that induces                                                      ID NO. sequence residues 0.5 maximum FI                                     ______________________________________                                        52     QTASSSSTL     37-45    >100   μg/ml                                   53 QTASSSSTLV 37-46 >100 μg/ml                                             1 STLVEVTLGEV 43-53 45 μg/ml                                               54 VTLGEVPAA 48-56 >100 μg/ml                                              55 VTKAEMLESV 130-139 70 μg/ml                                             56 VTKAEMLESVL 130-140 >100 μg/ml                                          57 VTCLGLSYDGL 179-189 >100 μg/ml                                          58 KTGLLIIVL 198-206 65 μg/ml                                              59 KTGLLIIVLA 198-207 80 μg/ml                                             60 KTGLLIIVLAI 198-208 >100 μg/ml                                          61 HTLKIGGEPHI 289-299 >100 μg/ml                                        ______________________________________                                    

                  TABLE III                                                       ______________________________________                                        Peptides derived from melanoma protein MAGE-2 binding to HLA-A2.1               Peptide    Amino acid             SEQ                                                                            No. sequence region ID NO                ______________________________________                                        1        STLVEVTLGEV  residues 43-53                                                                            1                                             -- LVEVTLGEV residues 45-53 2                                                 2 KMVELVHFL residues 112-120 3                                                3 VIFSKASEYL residues 149-158 4                                               4 YLQLVFGIEV residues 157-166 5                                               5 QLVFGIEVV residues 159-167 6                                                6 QLVFGIEVVEV residues 159-169 7                                              7 IIVLAIIAI residues 203-211 8                                                8 KIWEELSMLEV residues 220-230 9                                              9 ALIETSYVKV residues 277-286 10                                              10  LIETSYVKV residues 278-286 11                                           ______________________________________                                    

Most HLA-A2.1 binding peptides were found using the HLA-A2.1 motif, inaccordance with Falk et al., Nature 351: 290-296 (1991); Hunt et al.,Science 255: 1261-1263 (1992); and Nijman et al., J. Immunotherapy 14:121-126 (1993) . Only one additional HLA-A2.1 binding peptide was foundusing the extended HLA-A2.1 motif of Ruppert et al., Cell 74: 929-937(1993).

EXAMPLE 3

This example shows the in vitro induction of primary immune responses.As an illustration for the possibility of inducing primary responses ingeneral, including MAGE-2 peptides, such responses against HPV peptidesusing the processing defective cell line 174CEM.T2 are shown.

The expression of HLA-A2.1 on 174CEM.T2 cells (T2) is increased byincubating T2 cells in medium containing relevant peptide. T2 cells willpresent the relevant peptide bound to HLA-A2.1 in high amount andtherefore are good antigen presenting cells (APC). In the responseinducing method described recently (Kast et al., J. Immunotherapy 14:115-120 (1993) the T2 cell line is used as APC and post-Ficollmononuclear cells are used as responder cells.

Method

1) Peptide Loading of HLA-A2.1 on T2

T2 cells in a concentration of 2×10⁶ cells per ml were incubated for 13h at 37° C. in a T 25 flask (Becton Dickinson, Falcon, Plymouth England)in serum-free IMDM (=Iscoves Modified Dulbecco's Medium: Biochrom KG,Seromed Berlin, Germany,) with glutamine (2 mM, ICN Biochemicals Inc.,Costa Meisa, USA,), antibiotics (100 IU/ml penicillin (Brocades Pharma,Leiderdorp, The Netherlands, 100 μg/ml kanamycin (Sigma, St. Louis,USA,)) and the selected peptide from HPV MLDLQPETT (SEQ ID NO: 12 in aconcentration of 80 μg/ml.

2) Mitomycin C Treatment of T2 Antigen Presenting Cells (APC)

These incubated T2 cells were spun down and subsequently treated in adensity of 20×10⁶ cells/ml with Mitomycin C (50 μg/ml) in serum-freeRPMI (Gibco Paisley, Scotland,) medium for 1 h at 37° C. Hereafter theT2 cells were washed three times in RPMI.

3) Preparing for Primary Immune Response Induction

All wells of a 96-well-U-bottom plate (Costar, Cambridge, USA,) werefilled with 100,000 Mitomycin C-treated T2 cells in 50 μl serum-free,complete RPMI medium (glutamine (2 mM, ICN Biochemicals Inc., CostaMeisa, USA,), penicillin (100 IU/ml, Brocades Pharma, Leiderdorp, TheNetherlands), kanamycin (100 μg/ml, Sigma, St. Louis, USA,)) and thepeptide MLDLQPETT (SEQ ID NO: 12) in a concentration of 80 μg/ml;

4) Responder Cells

Responder cells are mononuclear peripheral blood lymphocytes (PBL) of aHLA-A2.1 subtyped donor (=C.B.). The PBL were separated from a buffycoat by Ficoll-procedure (Ficoll preparation: Lymphoprep ofNycomed-pharma, Oslo, Norway,) and washed twice in RPMI. Afterseparation and washing, the PBL were resuspended in complete RPMI mediumwith 30% human pooled serum (HPS) (HPS is tested for suppressionactivity in mixed lymphocyte cultures).

5) Incubation of Primary Immune Response

400,000 PBL-C.B. in 50 μl of medium (the medium described in paragraph4, supra were added to each well of the 96-well-U-bottom plate alreadyfilled with T2 cells and cultured for 7 days at 37° C. in an incubatorwith 5% CO₂ and 90% humidity.

6) Restimulation (day 7)

On day 7 after incubation of PBLS, peptide MLDLQPETT (SEQ ID NO:62) andT2 cells described supra, the PBLs were restimulated with peptideMLDLQPETT (SEQ ID NO:62). For this purpose all cells and medium out ofthe 96 wells were harvested. Viable cells were isolated by theFicoll-procedure and washed in RPMI. In a new 96-well-U-bottom plate50,000 of these viable cells were seeded in each well together with 50μl complete RPMI medium with 15% EPS. Per well 20,000 autologous,irradiated (3000 rad) PBLs and 50,000 autologous, irradiated (10000 rad)EBV-transformed B-lymphocytes (=EBV-C.B.) were added together with 50 μlof complete RPMI medium with 15% HPS and peptide MLDLQPETT (SEQ ID NO:62) in a concentration of 80 μg/ml. The cells were cultured for 7 daysat 37° C. in an incubator with 5% CO₂ and 90% humidity.

7) Restimulation (day 14)

On day 14 after incubation of PBLs, peptide MLDLQPETT (SEQ ID NO:62) andT2 cells, the PBLs were restimulated with peptide MLDLQPETT (SEQ IDNO:62). To do so the procedure for restimulation, Supra is repeated.

8) Cloning by Limiting Dilution

On day 21 after incubation of PBLs, peptide MLDLQPETT (SEQ ID NO:62) andT2 cells, cells and medium out of the 96 wells were harvested. Viablecells were isolated by the Ficoll-procedure and washed in complete RPMIwith 15% EPS. This bulk culture of viable cells was cloned by theLimiting Dilution. Into each well of a new 96-well-U-bottom plate(Costar, Cambridge, USA, cat. nr. 3799) 50 μl complete RPMI medium with15 % HPS was added together with 100 viable cells (=HPV16 bulk antiMLDLQPETT (SEQ ID NO:62)). For other new 96-well-U-bottom plates thiswas exactly repeated except for the number of cells for wells:subsequent plates contained dilutions of cells at 10, 1, or 0.3 cellsper well. To all wells 20,000 pooled and irradiated (3000 rad) PBL offour different donors and 10,000 pooled and irradiated (10,000 rad)EBV-transformed B-cells of three different HLA-A2.1 donors (VU-4/518/JY)were added together with 50 μl of complete RPMI medium with 15% HPS andpeptide MLDLQPETT (SEQ ID NO:62) in a concentration of 40 μg/ml,Leucoagglutinin in a concentration of 2% (Pharmacia, Uppsala, Sweden,),human recombinant IL-2 in a concentration of 120 IU/mL (Eurocetus,Amsterdam, The Netherlands).

9) Expand Clones

Add per well, in a final volume of 100 μl=>

25,000 viable cells

20,000 irradiated PBL-pool (described supra)

10,000 irradiated EBV-pool (described supra)

2 μg peptide MLDLQPETT (SEQ ID NO:62)

6 IU recombinant IL-2.

On day 49 a cytotoxicity assay was performed with 65 clones and one bulksample as effector cells and T2 (with or without the peptide MLDLQPETT(SEQ ID NO:62) as target cells. Background killing is defined as killingof T2 cells incubated with an irrelevant (but HLA-A2.1 binding) peptide:GILGFVFTL (SEQ ID NO:64). This influenza matrix protein-derived peptideis the epitope for HLA-A2.1 restricted influenza specific CTL and isknown in the art. The HPV bulk (C.B.) anti MLDLQPETT effector cellsseemed to be specific for killing MLDLQPETT sensitized cells.

A limiting dilution assay was done with the HPV bulk cells and, after 23days, a cytotoxicity assay was performed with five clones. Results of arepresentative clone is shown in FIG. 2.

The data suggest that the peptides of (SEQ ID NOS: 1-11 singlepolypeptides of identified sequences. However, homologs, isoforms orgenetic variants of these peptides may exist within or outside thecellular environment. This invention encompasses all such homologs,isoforms or genetic variants of the above peptides provided that theybind to the HLA-A2.1 molecule.

Polypeptides that are homologs of the peptides specifically includethose having amino acid sequences which are at least about 40% conservedin relation to the amino acid sequence set forth in Table II,preferentially at least about 60% conserved, and more preferentially atleast about 75% conserved.

It will be understood by one of ordinary skill in the art that othervariants of the peptides shown above are included within the scope ofthe present invention. This particularly includes any variants thatdiffer from the above mentioned and synthesized peptides only byconservative amino acid substitution. In particular, replacements of C(cysteine) by A (alanine), S (serine), α-aminobutyric acid and othersare included as it is known that cysteine-containing peptides aresusceptible to (air) oxidation during synthesis and handling. Many suchconservative amino acid substitutions are set forth as sets by Taylor J.Mol. Biol. 188: 233-258 (1986).

Herein the peptides shown above or fragments thereof include anyvariation in the amino acid sequence, whether by conservative amino acidsubstitution, deletion, or other processes, provided that thepolypeptides bind to the HLA-A2.1 molecule. The fragments of thepeptides may be small peptides with sequences of as little as five ormore amino acids, said sequences being those disclosed in Table II whensaid polypeptides bind to the HLA-A2.1 molecule.

Polypeptides larger than the peptides shown are especially includedwithin the scope of the present invention when said polypeptides inducea MAGE-2-specific CTL response in HLA-A2.1 positive individuals andinclude a (partial) amino acid sequence as set forth in Table II, orconservative substitutions thereof. Such polypeptides may have a lengthof from 9 to 12, more preferably 9 to 11 or even 9 to 10 amino acids.

This invention includes the use of polypeptides generated by everymeans, whether genetic engineering, peptide synthesis with solid phasetechniques or others. The foregoing peptides may have various chemicalmodifications made at the terminal ends and still be within the scopethe present invention. Also other chemical modifications are possible,particularly cyclic and dimeric configurations. The term "derivatives"intends to cover all such modified peptides.

The polypeptides of the present invention find utility for theprophylaxis diagnosis and/or treatment or prevention of diseasesinvolving MAGE-2 expressing cells including melanoma cells and othercancer cells.

For all applications the peptides are administered in an immunogenicform. Since the peptides are relatively short, this may necessitateadmixture, complexing, combining, conjugation, or chemical binding withan imunogenicity conferring carrier material such as lipids or others orthe use of adjuvants.

The magnitude of a prophylactic or a therapeutic dose of polypeptides ofthis invention will, of course, vary with the group of patients (age,sex, weight, etcetera), the nature of the severity of the condition tobe treated, the particular polypeptide of this invention and its routeof administration. Any suitable route of administration may be employedto achieve an effective dosage of a polypeptide identified by thisinvention, as well as any dosage form well known in the art of pharmacy.In addition the polypeptides may also be administered by controlledrelease means and/or delivery devices. They may also be administered incombination with other active substances such as, in particular, T-cellactivating agents like interleukin-2 etc.

The peptides of this invention may also be useful for other purposes,such as diagnostic use. For example, they may be used to check whether avaccination with a peptide according to the invention has beensuccessful. This may be done in vitro by testing whether said peptide isable to activate T cells of the vaccinated person.

Other aspects of the invention will be clear to the skilled artisan, andneed not be repeated here.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES:  64                                         - -  - - (2) INFORMATION FOR SEQ ID NO: 1:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 1:                         - - Ser Thr Leu Val Glu Val Thr Leu Gly Glu Va - #l                          1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 2:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acid residues                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 2:                         - - Leu Val Glu Val Thr Leu Gly Glu Val                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO: 3:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acid residues                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 3:                         - - Lys Met Val Glu Leu Val His Phe Leu                                      1                5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO: 4:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 4:                         - - Val Ile Phe Ser Lys Ala Ser Glu Tyr Leu                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 5:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 5:                         - - Tyr Leu Gln Leu Val Phe Gly Ile Glu Val                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 6:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acid residues                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 6:                         - - Gln Leu Val Phe Gly Ile Glu Val Val                                      1                5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO: 7:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 7:                         - - Gln Leu Val Phe Gly Ile Glu Val Val Glu Va - #l                          1                5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 8:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acid residues                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 8:                         - - Ile Ile Val Leu Ala Ile Ile Ala Ile                                      1                5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO: 9:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 9:                         - - Lys Ile Trp Glu Glu Leu Ser Met Leu Glu Va - #l                          1                5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 10:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 10:                        - - Ala Leu Ile Glu Thr Ser Tyr Val Lys Val                                  1                5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 11:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acid residues                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 11:                        - - Leu Ile Glu Thr Ser Tyr Val Lys Val                                      1                5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO: 12:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 12:                        - - Gly Leu Glu Ala Arg Gly Glu Ala Leu Gly Le - #u                          1                5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 13:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acid residues                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 13:                        - - Gly Leu Glu Ala Arg Gly Glu Ala Leu                                      1                5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO: 14:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acid residues                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 14:                        - - Ala Leu Gly Leu Val Gly Ala Gln Ala                                      1                5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO: 15:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acid residues                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 15:                        - - Gly Leu Val Gly Ala Gln Ala Pro Ala                                      1                5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO: 16:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acid residues                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 16:                        - - Asp Leu Glu Ser Glu Phe Gln Ala Ala                                      1                5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO: 17:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 17:                        - - Asp Leu Glu Ser Glu Phe Gln Ala Ala Ile                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 18:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 18:                        - - Ala Ile Ser Arg Lys Met Val Glu Leu Val                                  1                 5 - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 19:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acid residues                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 19:                        - - Ala Ile Ser Arg Lys Met Val Glu Leu                                      1                5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO: 20:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 20:                        - - Lys Met Val Glu Leu Val His Phe Leu Leu                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 21:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 21:                        - - Lys Met Val Glu Leu Val His Phe Leu Leu Le - #u                          1                5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 22:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 22:                        - - Leu Leu Leu Lys Tyr Arg Ala Arg Glu Pro Va - #l                          1                5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 23:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 23:                        - - Leu Leu Lys Tyr Arg Ala Arg Glu Pro Val                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 24:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 24:                        - - Val Leu Arg Asn Cys Gln Asp Phe Phe Pro Va - #l                          1                 5 - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 25:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 25:                        - - Tyr Leu Gln Leu Val Phe Gly Ile Glu Val Va - #l                          1                5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 26:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 26:                        - - Gly Ile Glu Val Val Glu Val Val Pro Ile                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 27:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acid residues                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 27:                        - - Pro Ile Ser His Leu Tyr Ile Leu Val                                      1                5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO: 28:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acid residues                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 28:                        - - His Leu Tyr Ile Leu Val Thr Cys Leu                                      1                5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO: 29:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 29:                        - - His Leu Tyr Ile Leu Val Thr Cys Leu Gly Le - #u                          1                5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 30:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acid residues                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 30:                        - - Tyr Ile Leu Val Thr Cys Leu Gly Leu                                      1                5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO: 31:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acid residues                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 31:                        - - Cys Leu Gly Leu Ser Tyr Asp Gly Leu                                      1                5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO: 32:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 32:                        - - Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu                                  1                5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 33:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acid residues                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 33:                        - - Val Met Pro Lys Thr Gly Leu Leu Ile                                      1                 5                                                            - -  - - (2) INFORMATION FOR SEQ ID NO: 34:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 34:                        - - Val Met Pro Lys Thr Gly Leu Leu Ile Ile                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 35:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 35:                        - - Val Met Pro Lys Thr Gly Leu Leu Ile Ile Va - #l                          1                5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 36:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acid residues                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 36:                        - - Gly Leu Leu Ile Ile Val Leu Ala Ile                                      1                 5                                                            - -  - - (2) INFORMATION FOR SEQ ID NO: 37:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 37:                        - - Gly Leu Leu Ile Ile Val Leu Ala Ile Ile                                  1                5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 38:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 38:                        - - Gly Leu Leu Ile Ile Val Leu Ala Ile Ile Al - #a                          1                5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 39:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acid residues                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 39:                        - - Leu Leu Ile Ile Val Leu Ala Ile Ile                                      1                5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO: 40:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 40:                        - - Leu Leu Ile Ile Val Leu Ala Ile Ile Ala  - #                  - #         1                                                                           5                  - # 10                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 41:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 41:                        - - Leu Leu Ile Ile Val Leu Ala Ile Ile Ala Il - #e                          1                5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 42:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acid residues                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 42:                        - - Leu Ile Ile Val Leu Ala Ile Ile Ala                                      1                5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO: 43:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 43:                        - - Leu Ile Ile Val Leu Ala Ile Ile Ala Ile  - #                  - #         1                                                                            5                  - # 10                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 44:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acid residues                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 44:                        - - Ile Ile Ala Ile Glu Gly Asp Cys Ala                                      1                5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO: 45:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acid residues                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 45:                        - - Lys Ile Trp Glu Glu Leu Ser Met Leu                                      1                5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO: 46:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 46:                        - - Leu Met Gln Asp Leu Val Gln Glu Asn Tyr Le - #u                          1                5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 47:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acid residues                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 47:                        - - Phe Leu Trp Gly Pro Arg Ala Leu Ile                                      1                5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO: 48:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acid residues                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 48:                        - - Leu Ile Glu Thr Ser Tyr Val Lys Val                                      1                5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO: 49:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 49:                        - - Ala Leu Ile Glu Thr Ser Tyr Val Lys Val Le - #u                          1                5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 50:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 50:                        - - Thr Leu Lys Ile Gly Gly Glu Pro His Ile                                  1                5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 51:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 51:                        - - His Ile Ser Tyr Pro Pro Leu His Glu Arg Al - #a                          1                5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 52:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acid residues                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 52:                        - - Gln Thr Ala Ser Ser Ser Ser Thr Leu                                      1                5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO: 53:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 53:                        - - Gln Thr Ala Ser Ser Ser Ser Thr Leu Val                                  1                5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 54:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acid residues                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 54:                        - - Val Thr Leu Gly Glu Val Pro Ala Ala                                      1                5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO: 55:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 55:                        - - Val Thr Lys Ala Glu Met Leu Glu Ser Val                                  1                5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 56:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 56:                        - - Val Thr Lys Ala Glu Met Leu Glu Ser Val Le - #u                          1                5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 57:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 57:                        - - Val Thr Cys Leu Gly Leu Ser Tyr Asp Gly Le - #u                          1                5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 58:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acid residues                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 58:                        - - Lys Thr Gly Leu Leu Ile Ile Val Leu                                      1                5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO: 59:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 59:                        - - Lys Thr Gly Leu Leu Ile Ile Val Leu Ala                                  1                5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 60:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 60:                        - - Lys Thr Gly Leu Leu Ile Ile Val Leu Ala Il - #e                          1                5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 61:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 61:                        - - His Thr Leu Lys Ile Gly Gly Glu Pro His Il - #e                          1                5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 62:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acid residues                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 62:                        - - Met Leu Asp Leu Gln Pro Glu Thr Thr                                      1                5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO: 63:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acid residues                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 63:                        - - Leu Ile Glu Thr Ser Tyr Val Lys Val Leu                                  5                  - # 10                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 64:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acid residues                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 64:                        - - Gly Ile Leu Gly Phe Val Phe Thr Leu                                     __________________________________________________________________________

We claim:
 1. Isolated peptide selected from the group consisting of SEQID SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ IDNO:
 10. 2. The isolated peptide of claim 1, designated SEQ ID NO:
 6. 3.Isolated cytolytic T cell clone specific for a complex of HLA-A2 and apeptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10and SEQ ID NO:
 11. 4. The isolated cytolytic T cell clone of claim 3,wherein said peptide is SEQ ID NO:
 1. 5. The isolated cytolytic T cellclone of claim 3, wherein said peptide is SEQ ID NO:
 6. 6. Method fortreating a subject with a cancerous condition in which cancer cellspresent a complex of HLA-A2 and a peptide molecule selected from SEQ IDNO: 1,2,3,4,5,6,7,8,9,10 and 11 on their surfaces, comprisingadministering an amount of an isolated cytolytic T cell clone specificfor said complex to said subject, sufficient to lyse said cancer cells.7. The method of claim 6, wherein said peptide is SEQ ID NO:
 1. 8. Themethod of claim 6, wherein said peptide is SEQ ID NO:
 6. 9. The methodof claim 6, wherein said peptide is SEQ ID NO: 9.